KARP-1: a novel leucine zipper protein expressed from the Ku86 autoantigen locus is implicated in the control of DNA-dependent protein kinase activity.

The Ku autoantigen plays an integral role in mammalian DNA double-strand break repair as the DNA binding component of the DNA-dependent protein kinase (DNA-PK) complex. Here, we demonstrate that a second gene, KARP-1 (Ku86 Autoantigen Related Protein-1), is expressed from the Ku86 locus. The KARP-1 gene utilizes an upstream promoter and additional exons which results in an extra 9 kDa of protein appended onto the normal Ku86 polypeptide. The KARP-1-specific domain encodes interdigitating hexa- and penta-heptad repeats of leucine residues flanked by a very basic region. Intriguingly, the catalytic subunit of DNA-PK also contains a hexa-heptad repeat of leucines. Consistent with this observation, we observed that human cell lines stably expressing dominant-negative constructs of KARP-1 resulted in diminished DNA-PK activity and X-ray hypersensitivity and that a KARP-1 antibody significantly neutralized DNA-PK activity in vitro. Finally, we present data which suggests that KARP-1 may be primate-specific. These observations have important repercussions for mammalian DNA double-strand break repair.     

The lethality of Ku86 (XRCC5) loss-of-function mutations in human cells is independent of p53 (TP53)

Ku86 is one of the two regulatory subunits of the DNA-PK (DNA-dependent protein kinase) complex that is required for DNA double-strand break repair in mammalian cells. In a previous study, by means of somatic gene targeting, we generated human cell lines deficient in Ku86 (XRCC5). Heterozygous human Ku86 cells exhibited a wide array of haploinsufficient phenotypes, including sensitivity to ionizing radiation, defects in DNA-PK and DNA end-binding activities, elevated levels of p53 (TP53) and gamma-H2AX foci, and a defect in cell proliferation with an increase in the frequency of aneuploid cells. Here we demonstrate that the overexpression of a human Ku86 cDNA complemented the deficiencies of these cells to wild-type levels. In contrast, Ku86 overexpression only partially rescued the telomere defects characteristic of Ku86 heterozygous cells and did not rescue their genetic instability. Additionally, in stark contrast to every other species described to date, we had shown earlier that homozygous human Ku86(-/-) cells are inviable, because they undergo 8 to 10 rounds of cell division before succumbing to apoptosis. The tumor suppressor protein p53 regulates the DNA damage response in mammalian cells and triggers apoptosis in the face of excessive DNA damage. Correspondingly, ablation of p53 expression has repeatedly been shown to significantly ameliorate the pathological effects of loss-of-function mutations for a large number of DNA repair genes. Surprisingly, however, even in a p53-null genetic background, the absence of Ku86 proved lethal. Thus the gene encoding Ku86 (XRCC5) is an essential gene in human somatic cells, and its absence cannot be suppressed by the loss of p53 function. These results suggest that Ku86 performs an essential role in telomere maintenance in human cells.     

Molecular cloning and characterization of rKAB1, which interacts with KARP-1, localizes in the nucleus and protects cells against oxidative death

The Ku autoantigen/KARP-1 (Ku86 autoantigen related protein-1) plays an important role in the double-strand break repair of mammalian DNA as a DNA-binding component of DNA-dependent protein kinase (DNA-PK) complex. KARP-1 is differently transcribed from the human Ku86 autoantigen gene locus and it is implicated in the control of DNA-dependent protein kinase activity. We cloned rKAB1, a rat homolog of KAB1 (KARP-1 binding protein 1 of human) from a rat hippocampal cDNA library. rKAB1 mRNA was specifically expressed in the brain and the thymus. EGFP-tagged rKAB1 protein localized in cell nucleus and in the condensed chromosome during the mitotic cell division. We found that rKAB1 works as a protective protein against cell damage by oxidative stress.